Studies On The Function Of A New Demethylase Gene OsDML4 In Rice

Rice is a gramineous crop sensitive to temperature.High temperature(HT)can affect the accumulation of seed storage materials and cause adverse effects on the yield and quality of rice.As one of the most crucial epigenetic modifications,DNA methylation is vital for plant growth and development.However,the effect of temperature and DNA methylation interaction on rice seed development has not been deeply studied yet.In previous study,we identified a new demethylase gene OsDML4.In order to explore the molecular function of OsDML4,especially its role in rice seed development and storage materials accumulation,we use CRISPR-Cas9 engineering technology to generate osdml4 mutants.The osdml4 mutants showed different phenotypes under HT and NT.The content of starch and SSPs were detected and the morphology and structure of starch granules and PBs were observed and analyzed.RNA-Seq,WGBS and related molecular experiments were used to explore the effects of mutation of OsDML4 on the whole genome DNA methylation and the expression of seed storage materials biosynthesis genes and their important regulatory factors to clarify how OsDML4 regulates rice growth and development through DNA methylation under different temperatures.The main results are as follows:1.Knockout of OsDML4 can induce floury endosperm under HT.The content of total starch and amylose was decreased in the osdml4 mutants.The starch granules of the osdml4 floury endosperm were irregularly round and loosely arranged.The seed length of the osdml4 mutants was significantly longer than those of WT,while the thicknesses and 1000-grain weight were considerably reduced.Furthermore,knockout of OsDML4 increased the total protein content,but dramatically reduced the accumulation of glutelins and 16-k Da prolamin.The absence of OsDML4 also caused adverse effects on the formation of PBs,the number of PB-II was greatly decreased and incomplete PB-II with empty space and abnormally shaped PB-II were observed in the osdml4 mutants.However,there was no significant difference between the osdml4 mutants and WT under NT,indicating OsDML4 involved in high temperature induced floury endosperm formation in rice.RNA-Seq analysis of 15 DAP endosperm of WT and the osdml4 mutant showed that OsDML4 could participate in many biological processes and metabolic pathways,such as seed maturation,germination and dormancy,nutrient reservoir activity,biosynthesis and metabolism of many storage compounds,protein storage vacuole,gene transcription and regulation of transcription.Knockout of OsDML4 significantly decreased the transcription of glutelin and 16-k Da prolamin genes,as well as the expression of their important regulators RISBZ1、RPBF、VPE1、Bi P1、PDIL1-1 and most starch biosynthesis related genes under HT.In addition,knockout of OsDML4 can also change the expression of HSP and kinase related genes,indicating that OsDML4 may play an important role in stress response.2.Knockout of OsDML4 can significantly increase the DNA methylation in the whole genome.WGBS analysis of 15 DAP endosperm of WT and the osdml4 mutant revealed much higher global methylation levels of CG,CHG and CHH contexts in the osdml4 mutant compared to WT under HT.While under NT,CG methylation level in the osdml4 mutant was slightly lower than that of WT,and CHG and CHH methylation levels were slightly increased in the osdml4 mutant compared to WT.The DNA methylation level of promoter and gene body regions increased significantly under HT,while under NT,only CHH methylation level was increased at the promoters,but the degree of increase was much less than that of HT.The number of hyper-DMRs identified under HT was much more than that of NT.The analysis of DNA methylation level in the promoter region of genes related to storage materials showed that knockout of OsDML4 only increased the DNA methylation level of the promoters of RISBZ1,SSIIc,ISA2 and Os BT1 and the gene body region of AGPS2 under HT,but the DNA methylation levels of the glutelin genes,RPBF,VPE1,Bi P1 and PDIL1-1 promoters were not affected under both HT and NT,suggesting that the demethylation function of OsDML4 also depends on temperature.OsDML4-mediated demethylation directly affects the expression of some storage materials related genes and participates in the formation of floury endosperm.In conclusion,our study demonstrates a novel OsDML4-mediated epigenetic regulatory mechanism involving in the formation of floury endosperm,which enriches the functional research and knowledge system of rice demethylase genes,provides a new perspective in the formation of floury endosperm and accelerates the process of epigenetics participating in rice molecular breeding.